Important note: Recently, several oncology centers started pre-hydration of the patient with 1 Lit
saline over 1-2h prior to Lipoplatin administration. Prehydration
can shorten the time of the subsequent Lipoplatin infusion from 8 h down to 2-3
hours and it reduces nephrotoxicity and bone-marrow
toxicity of Lipoplatin. Lipoplatin (about 2 vials, 300 mg total in encapsulated
cisplatin giving for a patient of 1.5 m2
body surface area a dose of 200 mg/m2) can be administered subsequently
in 1 Lit 5% dextrose or saline (dextrose is preferred to open the appetite of
cancer cells for nutrients). For patients who cannot receive large volumes of
saline or 5% dextrose, a slower infusion of the drug in smaller dilution (e.g.,
500 ml) is recommended. A post-hydration with 1 Lit saline is also recommended.
Patients should also receive as premedication 4 mg dexamethasone, antiemetics (ondansetron)
and anti-histamine treatment (Benadryl, Generic Name: diphenhydramine) to
reduce the chance of allergies from liposomes.

IMPORTANT: Lipoplatin monotherapy
on Day 1 using 200 mg/m2 Lipoplatin in combination with low-dose
adjuvant radiation on Day 2 with 2 Gy dose every week
for 6-9 weeks is our established treatment regimen for an optimal treatment
that is void of side effects and can be taken for a long period without
cumulative toxicity. This is followed by maintenance treatment once every 3-4
weeks depending on the severity and confinement of the disease. It has no side
effects (only mild Grade 1) and can treat almost all cancers tested (breast, glioblastoma, lung, pancreatic, colorectal). Because of the
absence of side effects it is also recommended for all other solid tumors but
also for leukemias (Lipoplatin targets bone marrow)
and pediatric tumors. Such a treatment will de-bulk the tumor mass and the residual
disease can be treated with immunotherapy or surgery to eradicate cancer.

Table of Contents

1.
NAME OF THE MEDICINAL PRODUCT

2.
QUALITATIVE AND QUANTITATIVE COMPOSITION

3.
PHARMACEUTICAL FORM

4.
CLINICAL PARTICULARS

4.1
Therapeutic indications

4.2
Posology and method of administration

4.3
Contraindications

4.4
Special warnings and precautions for use

4.5
Interaction with other medicinal products and other forms of interaction

4.6
Pregnancy and lactation

4.7
Effects on ability to drive and use machines

4.8
Undesirable effects

4.9
Overdose

5.
PHARMACOLOGICAL PROPERTIES

5.1.
Pharmacodynamic properties

5.2
Pharmacokinetic properties

5.3
Preclinical safety data

5.4
Mechanism of action of Lipoplatin

5.4.1
Molecular Mechanisms of Cisplatin and Lipoplatin for
Entry into Tissues and Cells

The drug
substance cisplatin has a concentration of 3mg/ml
within the liposomal formulation, therefore a 50ml
vial contains 150mg of cisplatin.

Lipoplatin is
a proprietary liposomally-encapsulated drug product of the FDA-approved,
commercially-available cytotoxic agent cisplatin. In
the Lipoplatin product, cisplatin (cis-diaminodichloroplatinum) is encapsulated in a liposome
shell composed of dipalmitoylphosphatidyl
glycerol, soy phosphatidyl choline, cholesterol and methoxy-polyethylene
glycol-distearoylphosphatidylethanolamine
lipid conjugate. The ratio of cisplatin to lipids is
8.9%:91.1% (w/w).

For the
Excipients please refer to paragraph 6.1.

3. PHARMACEUTICAL FORM

Concentrate
nanoparticle suspension for infusion.

150 mg in cisplatin for both Lipoplatin and Nanoplatin
in a glass vial of 50-ml

4. CLINICAL PARTICULARS

4.1. Therapeutic indications

Lipoplatin is
used against pancreatic cancer in combination with gemcitabine as first
line treatment in a pivotal EMA study. Nanoplatin is
used against non-squamous non-small cell lung cancer (ns-NSCLC) as first
line treatment in combination with pemetrexed in a
pivotal EMA study. A Phase II in NSCLC has been completed (Stathopoulos et al 2010 Liposomal cisplatin
combined with paclitaxel versus cisplatin and
paclitaxel in non-small-cell lung cancer: a randomized phase III multicenter
trial. Ann Oncol 21:2227-32). Also a Phase III
on non-squamous NSCLC has been completed (Stathopoulos
et al 2011 Comparison of liposomal cisplatin
versus cisplatin in non-squamous cell non-small-cell
lung cancer. Cancer ChemotherPharmacol
68:945-950). As an investigational drug Lipoplatin is recommended
against metastatic breast cancer in combination with vinorelbine
as first line treatment (Farhat et al 2011 A Phase II Study of Lipoplatin
(Liposomal Cisplatin)/Vinorelbine
Combination in HER-2/neu-Negative Metastatic Breast
Cancer. Clin Breast Cancer 11:384-9). As an
investigational drug Lipoplatin is used against gastric cancer in
combination with low dose radiation therapy and 5-FU (Koukourakis et al
2010 Concurrent liposomal cisplatin (Lipoplatin),
5-fluorouracil and radiotherapy for the treatment of locally advanced gastric
cancer: a phase I/II study. Int J RadiatOncolBiolPhys 78:150-5). Lipoplatin is
suggested to be used in all indications where cisplatin
has applications notably in metastatic testicular tumours,
non-seminomatous germ cell carcinoma, advanced stage
and refractory ovarian carcinoma, advanced stages and refractory bladder
carcinoma and squamous cell carcinoma of head and neck.

4.2. Premedication, Posology and method of administration

The first vial
of Lipoplatin of 150 mg (50 ml) is diluted in 1Lit 5% dextrose and is
administered intravenously for 2.5 h. Each of the subsequent vials of
Lipoplatin of 150 mg (50 ml) is diluted in 1 Lit saline and is administered
intravenously for 2.5 h. The recommended dose of Lipoplatin is 200 mg/m2
every 7 days or 200 mg/m2 D1,2 every 14
days as monotherapy (giving twice the dose of Lioplatin in combination therapies). The recommended
dose in combination chemotherapy (with gemcitabine, vinorelbine,
Paclitaxel ot other drugs) is 200 mg/m2 D1,8 in a 21-day schedule. The slow administration, especially
in the beginning of the drug infusion, is of great importance to assess allergy
or epigastric pain in patients. The slow
administration along with high volume of 5% dextrose or saline is of great
importance in establishing the low renal and gastrointestinal toxicities of the
drug.

Lipoplatin is
administered in doses dependent on the calculated body surface area. Body
weight measurements and appropriate dose adjustments will be made before each
infusion.

Drug remaining
in a vial after an infusion may be stored at 0 to 4 degrees Celsius and used in
a following infusion.

Batch number
of the drug used in every infusion should be recorded in patient's hospital
files.

Lipoplatin 3
mg/ml Sterile Concentrate is diluted in 1Lit 5% Dextrose for the first vial and
into 1 Lit Saline for all subsequent vials. The long infusion time of 5 to 8
hours is known to decrease the gastrointestinal and renal toxicities of
Lipoplatin.

4.3. Contraindications

Use is
contraindicated in those patients with a history of allergic reaction to cisplatin or to other platinum containing compounds. Use is
contraindicated in those patients (less than 1%) who develop an allergic
reaction to liposomes during the infusion.

Although cisplatin induces nephrotoxicity, which is cumulative and
is contraindicated in patients with renal impairment, Lipoplatin has been
administered without further renal impairment to such patients. A study on 40
cancer patients with renal failure has demonstrated safety in Lipoplatin plus
gemcitabine administration (Stathopoulos et al, 2011
J ClinOncol 29: suppl; abstr 7072).

Although cisplatin has been shown to cause cumulative ototoxicity
(which is reduced with pre- and post-hydration) and is contraindicated to
patients with hearing impairment, Lipoplatin has a negligible ototoxicity
profile.

Although cisplatin has bone marrow toxicity (which is reduced with
pre- and post-hydration) and is contraindicated in myelosuppressed
patients, Lipoplatin has a milder bone marrow
toxicity; never-the-less Lipoplatin should be used with caution to such
patients.

4.4. Special warnings and precautions for use

Lipoplatin
should only be administered under the supervision of oncologists in specialist
units under conditions permitting adequate monitoring and surveillance.
Supportive equipment should be available to control anaphylactic reactions.

Cisplatin reacts with metallic aluminium to form
a black precipitate of platinum. Although cisplatin
is protected from the lipid shell in the liposome nanoparticle in its Lipoplatin/Nanoplatin
formulation, all aluminium containing i.v.sets, needles,
catheters and syringes should be avoided.

The solution
for infusion should not be mixed with other drugs or additives.

About 12-30%
of the patients experience an epigastric
pain during the first minutes of Lipoplatin infusion that lasts for 5-10
min and subsides without medication (Stathopoulos et
al, 2005Oncol Rep 13, 589-595).
Therefore, the drug should be infused slowly at the beginning in new patients
till their sensitivity to the product is established.

Because cisplatin produces cumulative nephrotoxicity
which may be potentiated by aminoglycoside antibiotics, caution should
be used for simultaneous treatment of a patient with Lipoplatin and
aminoglycoside antibiotics. Serum creatinine, plasma
urea or creatinine clearance and magnesium, sodium
potassium, and calcium levels should be measured prior to initiating therapy,
and prior to each subsequent course. In general, the lowering of cisplatin's nephrotoxicity by Lipoplatin has been
demonstrated in numerous studies (Stathopoulos et al, 2010
Ann Oncol, 21:2227-32. Mylonakis
et al, 2010 Lung Cancer. 68:240-247). A study on 40 cancer
patients with renal failure has demonstrated safety in Lipoplatin plus
gemcitabine administration (Stathopoulos et al, 2011
J ClinOncol 29: suppl; abstr 7072).

Anaphylactic-like reactions to cisplatin have been
reported; these reactions have occurred within minutes of administration to
patients with prior exposure to cisplatin and have
been alleviated by administration of adrenaline, steroids and antihistamines.
However, no reports so far show anaphylactic-like reactions to Lipoplatin.

Neurotoxicity appears to be cumulative during cisplatin
administration and prior to each course, the absence of symptoms of peripheral
neuropathy is recommended for cisplatin treatment.
However, Lipoplatin exerts a much lower neuropathy to patients (see Undesirable
Effects).

Peripheral
blood counts should be monitored at 7 days after each infusion.

During, and
for at least three months after therapy, both male and female patients should
take contraceptive measures. As is the case with all anticancer drugs,
in men this drug may cause transitional or permanent sterility. Preservation of
sperm may be considered for the purpose of later fatherhood (see section 4.6,
Pregnancy and lactation).

4.5 Interaction with other medicinal products and other
forms of interaction

Lipoplatin can
be used in combination with other cytostatics that
have their own pattern of side effects. The myelo- nephro-, oto- and
gastrointestinal- toxicities induced by cisplatin in
the Lipoplatin formulation, although much lower than those of cisplatin, will be additive to existent impairment.
Neutropenia and nephrotoxicity Grade 3 are the dose limiting toxicities for
Lipoplatin observed at a dose of 350 mg/m2 in monotherapy
whereas alopecia and neurotoxicity are the main dose limiting toxicities observed with Lipoplatin 250 mg/m2 and
paclitaxel 175 mg/m2 (Stathopoulos et al 2010
Liposomal cisplatin dose escalation for determining
the maximum tolerated dose and dose-limiting toxicity: a phase I study.
Anticancer Res. 30:1317-1321).

4.6 Fertility, pregnancy and lactation

Use in Pregnancy:

Cisplatin has been shown to be mutagenic in bacteria and is teratogenic
and embryotoxic in mice. Cisplatin
should not be used during pregnancy unless the clinician considers the risk in
an individual patient to be clinically justified. Similarly, Lipoplatin should
not be used during pregnancy unless the clinician considers the risk in an
individual patient to be clinically justified. As with most chemotherapy drugs,
fertile female patients should have a recent negative pregnancy test prior to
admission to the protocol for Lipoplatin treatment.

Male and
female patients should take contraceptive measures during, and for at least
three months after therapy.

Use in Lactation:

Lipoplatin
should not normally be administered to mothers who are breastfeeding.

4.7 Effects on ability to drive and use machines

There are no
known effects of Lipoplatin on the ability to drive or operate machinery in
treated patients. However the profile of undesirable effects (central nervous
system and special senses) may reduce the patients' driving skills and
abilities to operate machinery.

4.8 Undesirable effects

Phase I
patients did not present any nephro-, oto- or neurotoxicity. The only reported adverse events
were grade 1-2 nausea and vomiting, and myelotoxicity.

A mild,
transient increase in hepatic enzymes may be seen in some patients. However,
there is no concern with the hepatotoxicity of Lipoplatin as monotherapy. Special precautions should be used when using
a combination drug known to cause hepatotoxicity.

Nephrotoxicity:

Grade 1-2 increase
in serum creatinine levels may be seen in single
infusions, especially in the first cycle of treatment. No grade 3-4 increase
has been reported, except in the case of rapid infusion of Lipoplatin (in 2-3
hours instead of 8 hours).

Mild nausea
and vomiting is a common adverse event (69% of patients). Grade 3 nausea and
vomiting is seen in less than 5% of patients.

Grade 1-2 diarrhoea is seen in 20% of patients.

Neurotoxicity:

Although
peripheral neuropathies and paresthesias in both
upper and lower extremities have been reported with cisplatin
administration that may occur after prolonged therapy or even after a single
dose and may progress after stopping treatment, neuropathies from Lipoplatin
treatment at doses of 120 mg/m2 every 7 days for 9 weeks are mild
and occur in a smaller percentage of patients (Mylonakis et al, Lung
Cancer. 2010 68:240-247).

Several
additional clinical studies have also established the lower neurotoxicity of LIpoplatin compared to cisplatin.

Hypersensitivity and allergic reactions:

Mild allergic
reactions (erythema, mild bronchospasm, tachycardia) are reported in
approximately 10% of patients, and relate to sensitivity to liposomes. In these
cases, corticosteroids +/- antihistamines are administered prior to the
following infusions.

Acute, severe epigastric/lumbar pain, lasting for approximately 5 minutes
and spontaneously subsiding, was seen in approximately 30% of the Phase I
patients. This adverse event is characteristic of liposomal drugs.

Although
anaphylactic-like reactions, possibly secondary to cisplatin
therapy, have been occasionally reported in patients previously exposed to cisplatin (rashes, urticaria,
erythema or pruritis) such anaphylactic-like
reactions have not been observed with Lipoplatin.

Electrolyte changes:

Electrolyte
changes, mainly hypomagnesaemia may be seen in a small percentage of patients.

Less common
adverse events are alopecia, neurotoxicity and metallic taste.

Toxicities are
less common when the rate of infusion is slow, as dictated in the study
protocol.

4.9. Overdose

Although acute
overdosage of cisplatin may
result in: [kidney failure, liver failure,
deafness, ocular toxicity including detachment of the retina, significant myelosuppression, intractable nausea and vomiting and death
from dysfunction of the respiratory centre with
acid/base imbalance from penetration to the blood-brain barrier] no
serious effects are anticipated with Lipoplatin overdose.

Overdosage can be expected to cause the toxic effects described above, but to an
exaggerated degree. Adequate hydration and osmotic diuresis may help reduce the
toxicity of cisplatin if administered promptly
following overdosage.

Although the
recommended dose is 200 mg/m2, patients have been treated with 350
mg/m2 that is defined as the MTD in Lipoplatin monotherapy.
At the 350 mg/m2 neutropenia Grade 3 and
nephrotoxicity Grade 3 were observed in 25% of the patients; however, there was
no Grade 4 toxicity of any kind (including nephrotoxicity) (Stathopoulos et al, 2010 Anticancer Res 30, 1317-1322).

Although cisplatin is administered for 4 to 6 cycles at its
recommended dose and further treatments are not used because of its cumulative
toxicity, Lipoplatin has been administered at up to 27 doses to a single
patient without severe cumulative toxicity. Thus, Lipoplatin can be used as a
maintenance therapy for extended periods.

5. PHARMACOLOGICAL PROPERTIES

5.1. Pharmacodynamic properties

Class of drug:
platinum drug or alkylating agent (FDA classification)

The ATC Code
for cisplatin is: LO1X A01 Platinum compound.

Lipoplatin
will receive its ATC code once approved for marketing authorization

Cisplatin has biochemical properties similar to that of bifunctional
alkylating agents producing inter-strand and intra-strand crosslinks in DNA.
New mechanisms of action for cisplatin have recently
been revealed including modulation of the mitochondrial, ERK and other signalling pathways depending on the cell type (reviewed by Boulikas 2007 Cancer Therapy 5, 349-376).
Although the principal mechanism of action of cisplatin
appears to be inhibition of DNA synthesis by crosslinking, other mechanisms,
including enhancement of tumor immunogenicity, may be involved in its
antineoplastic activity. Cisplatin also has immunosuppressive,
radiosensitising, and antimicrobial properties. Cisplatin does not appear to be cell cycle specific.

By extension
Lipoplatin also acts by DNA crosslinking but also by modulating signalling pathways, enhancement of tumor immunogenicity, radiosensitising activity and antimicrobial activity;
however, its mechanism of action has some additional characteristics,
supposedly from its membrane penetration. For example, the signalling
modulation signature profile is distinct from cisplatin's
in the same cell type. Also the cellular uptake of Lipoplatin™ and Lipoxal™, are higher in cell cultures compared to cisplatin and oxaliplatin
consistent with the proposed mechanism of fusion with the cell membrane (Tippayamontri et al 2011 Cellular uptake and cytoplasm / DNA
distribution of cisplatin and oxaliplatin
and their liposomal formulation in human colorectal cancer cell HCT116. Invest
New Drugs 29:1321-1327).

5.2 Pharmacokinetic properties

From a Phase I
study, total platinum concentrations measured in the diluted plasma samples and
free plasma concentrations measured in the plasma ultrafiltrates
were used for the pharmacokinetic analysis on 17 patients. During the 8 h
infusion period, the maximum platinum level in the plasma, attained at 6 h for
a dose of 125 mg/m2, was 5.7 g/ml and declined thereafter (Stathopoulos et al, 2005: Pharmacokinetics and
adverse reactions of a new liposomal cisplatin
(Lipoplatin): Phase I study Oncol Rep 13, 589-595).

Platinum
concentrations were also determined in surgical specimens at ~20h postinfusion in patients undergoing prescheduled surgery.
Platinum levels were the highest in the stomach tumors (40-200 μg/gr tissue)
compared to 4 μg/gr in normal stomach tissue. Liver tumors displayed 30-100 μg/gr tissue compared to 3-4 μg/gr in normal liver
tissue. Finally colon tumor specimens displayed 6-7 μg/gr
tissue compared to 0.02 μg/gr in normal colon tissue. In muscle tissue it was
zero and in fat tissue15-30 μg/gr tissue. A colon metastasis from
hepatocellular carcinoma displayed ~2 μg/gr tissue total platinum compared to
0.08 μg/gr in normal colon tissue from the same patient (Boulikas et al, 2005. Systemic Lipoplatin infusion
results in preferential tumor uptake in human studies. Anticancer Res,
25:3031-3040). Currently there are no data available on platinum levels
attained in lung, breast or other tumors but their platinum uptake has been
proposed to correlate to their degree of vascularization and maturation of the
wall of tumor vasculature.

Studies on
mice and rats have shown that the highest concentration of platinum after bolus
tail vain Lipoplatin or intraperitoneal
injection is in the kidney tissue but without causing nephrotoxicity to the
animals. After several days (also pronounced after repetitive administrations)
there is accumulation in liver and spleen again without impairment in the
hepatic function of the animals (Boulikas 2004
Low toxicity and anticancer activity of a novel liposomal cisplatin
(Lipoplatin) in mouse xenografts. Oncol Rep 12: 3-12).

After cisplatin intravenous administration, the clearance
of total platinum from plasma is rapid during the first four hours, but then
proceeds more slowly because of covalent binding to serum proteins. Levels of
unbound platinum fall with a half-life of 20 minutes to 1 hour depending on the
rate of drug infusion. On the contrary, after Lipoplatin intravenous
administration the half life is 50-116 hours
establishing the long circulation properties of the Lipoplatin nanoparticles.
The long circulation property of Lipoplatin nanoparticles is essential
for their extravasation to tumors through the compromised endothelium of their
vasculature.

The
elimination of intact cisplatin and various
platinum-containing biotransformation products is via the urine. About 15-25%
of administered platinum is rapidly excreted in the first 2-4 hours after
administration of cisplatin. This early excretion is
mostly of intact cisplatin. In the first 24 hours
after administration, 20-80% is excreted, the remainder representing drug bound
to tissues or plasma protein.

On the
contrary, after Lipoplatin intravenous administration, 9.1% was excreted
in the urine during the 8 h of i.v.infusion, 16.8% during the following 16 h and 10% during the
following 24 h. During the third day, an additional 4.8% was excreted in the
urine. Therefore, during the 3 days from the start of the Lipoplatin infusion
about 40.7% of total platinum is excreted in the urine (Stathopoulos et al, 2005: Pharmacokinetics and adverse reactions
of a new liposomal cisplatin (Lipoplatin): Phase I
study Oncol Rep 13, 589-595).

5.3 Preclinical safety data

Cisplatin has been shown to be mutagenic and may also have an anti-fertility
effect. The mutagenic and anti-fertility effects of Lipoplatin have not been
studied. However, animals can be administered with doses that are 5-7 times
higher than Cisplatin in order to start producing the
same side effects.

Preclinical studies have
shown the lower nephrotoxicity and other adverse effects of Lipoplatin,
compared to cisplatin, in mice, rats, and in SCID
mice (Boulikas 2004 Low toxicity and anticancer
activity of a novel liposomal cisplatin (Lipoplatin)
in mouse xenografts. Oncol Rep 12: 3-12.Devarajan et al 2004: Low renal toxicity of
Lipoplatin compared to cisplatin in animals.
Anticancer Res, 24, 2193-2200) and dogs with applications in veterinary
oncology (Marr et
al 2004 Preclinical evaluation of a liposome-encapsulated formulation of
cisplatin in clinically normal dogs. Am J Vet Res 65,
1474-1478). Studies in cell lines have deciphered one plausible
mechanism for the sensitivity of certain tumor cell lines to Lipoplatin (Fedier et al 2006 MLH1-deficient tumor cells are resistant
to lipoplatin, but retain sensitivity to lipoxal. Anticancer Drugs 17:315-23). Analysis of
molecular markers known to be related to cisplatin
resistance showed a direct correlation between cisplatin
and lipoplatin resistance and ERCC1 and LRP
expression and were proposed as valid predictors of sensitivity or resistance
to these drugs (Arienti et al 2008 Activity of lipoplatin
in tumor and in normal cells in vitro. Anticancer Drugs. 19:983-90).

A study in
rats has shown that Lipoplatin 12 mg/Kg has a lower neurotoxicity to cisplatin 4 mg/Kg after intraperitoneal
administration once weekly for 4 weeks. The onset of
peripheral neurotoxicity was assessed by measuring tail nerve conduction
velocity (NCV), morphological and morphometric analysis of dorsal root ganglia
(DRG), and morphological analysis of the sciatic nerve (Canta et al. 2011 Cancer ChemotherPharmacol. 68:1001-8).

5.4 Mechanism of action of Lipoplatin

5.4.1
Molecular Mechanisms of Cisplatin and Lipoplatin for
Entry into Tissues and Cells

Cisplatin (also carboplatin and oxaliplatin) are actively imported by cells using the Ctr1
(Copper transport protein 1) first identified in Saccharomyces cerevisiae and later across species. Ctr1 actively mediates
influx and intracellular accumulation of platinum in human cells. ATP7B
actively outfluxes platinum drugs from the cytoplasm
to the extracellular space. A major mechanism of resistance of tumor cells to
platinum drugs is associated with down regulation of Ctr1 or upregulation of ATP7B. An independent mechanism to platinum
drug resistance includes upregulation of DNA repair
mechanisms in resistant cells. Additional mechanisms are upregulation
of glutathione or metallothionein levels for cisplatin detoxification.

Lipoplatin on the other hand enters cells rapidly either via phagocytosis / endosomal engulfment or by the direct fusion of the
Lipoplatin nanoparticles with the cell membrane because of the presence of the fusogenic lipid DPPG on its shell. Lipoplatin accumulates
into cancer tissues supposedly by the extravasation of the nanoparticles
through the compromised endothelium sprouted during neoangiogenesis. Thus,
Lipoplatin concentration in normal tissues is minimized explaining its low
toxicity. A higher concentration of Lipoplatin in primary or metastatic tissue
compared to the adjacent normal tissue has been shown in surgical specimens in
patients at 20h from Lipoplatin infusion (Boulikas et
al, 2005 Anticancer Res. 25: 3031-3040).

Lipoplatin has
been shown to bypass resistance of cells, presumably at the cell membrane
level; indeed, previously cisplatin–treated
patients with NSCLC who relapsed, responded to Lipoplatin treatment with a
partial response of about 25% (Froudarakis et al, manuscript in preparation). This study shows
that Lipoplatin can treat tumors resistant to platinum drugs.

The scheme shows a blood vessel in
tumor tissue. Lipoplatin nanoparticlesof
100nm in diameter are depicted as spheres with the yellow toxic payload of cisplatin inside them. In normal tissue, blood vessels are
impenetrable by small nanoparticles. On the contrary, tumor blood vessels have
imperfections (tiny holes) in their walls (called endothelium); tumor blood
vessels are established during the process of neo-angiogenesis (meaning
sprouting of new blood vessels by a tumor cell mass during its growth phase).
Lipoplatin nanoparticles take advantage of these tiny holes to pass through and
extravasate inside the tumor reaching a concentration
that can be 10- to 200-fold higher compared to the
adjacent normal tissue.

Efficient
internalization of cisplatin in mammalian
cells has been shown to elicit generation of reactive oxygen species, which in
turn modulate signal transduction pathways that ultimately lead to cell death
by either apoptosis or necrosis. The two chloride ligands of cisplatin play a pivotal role in commencing cisplatin's toxic cascade. Indeed, due to the lower
chloride ion content of the cytoplasm, upon intracellular entry of cisplatin, both its chloride groups exchange with water to
yield diaquo (hydroxo) cisplatin species. These intermediates are highly reactive
against nucleophilic groups on either DNA or
proteins. For example, diaquo-cisplatin attacks on
DNA may lead to the formation of intra- or interstrand
DNA crosslinks, or to the formation of DNA monoadducts.
The major product is 1,2-interstrand d(GpG) crosslinks, although d(ApG)
crosslinks may also be observed. In other cases, only one cisplatinhydroxo species crosslinks DNA, while the other
attacks nucleophilic groups (thiols,
amino, hydroxyl) of proteins of various sizes, thereby forcing them to
crosslink on the DNA.

Efficient
access of hydroxo-cisplatin intermediates to DNA
depends on the structure and state of activation of chromatin. In quiescent
cells chromatin is highly condensed. In contrast, chromatin structure opens up
locally during either DNA replication or transcription. Relaxation of chromatin
structure leads to local melting of the nucleosome structure, which in turn
renders the linker regions of the nucleosome octamers
free to interact with factors from solution, such as promoter-binding
transcription factors as well as toxic hydroxo-cisplatin
intermediates. Cancer cells divide faster, replicate mutated DNA much more
frequently and relax key gene chromatin structure more often than normal cells;
this renders them significantly more susceptible to cisplatin
and hence Lipoplatin than normal cells.

Formation of
DNA adducts in cisplatin-treated cells alters the
pattern and/or binding affinity for many DNA-interacting protein complexes,
thereby triggering mechanisms of DNA repair, cell cycle arrest, apoptosis and
necrosis. Although the spatial and temporal orchestration of these events is
poorly understood, several putative effectors have been identified and proposed
to mediate downstream signaling in response to cisplatin-generated
DNA damage. For example, the activity of the high-mobility group box protein
HMGB1 may modulate the efficiency of nucleotide excision, mismatch repair and
chromatin remodeling in response to cisplatin.
Moreover, the tumor suppressor protein p53 may in turn regulate a plethora of
downstream genes and effectors in response to cisplatin
treatment. Finally, other effectors, such as the AKT and c-abl
kinases, as well as members of the Mitogen Activated Protein kinase superfamily
including the stress-responsive JNK and p38 kinases have also been implicated
in the signal transduction of cisplatin cytotoxicity.

Lipoplatin has similar molecular effects on cells. However, because of its tumor
accumulation and rapid entrance through the cell membrane to cytoplasm and nycleoplasm its effects on tumor cells including signalling cascades are amplified while minimizing damage
to normal tissue.

After cisplatin damage cells have two options: either to repair
the damage or undergo apoptosis. Tumor cells have developed mechanisms to evade
apoptotic death; one mechanism is accumulation of mutations in their DNA
allowing for clonal selection and expansion of those cells that have survival
advantages in a platinum drug environment forming resistant tumors. Lipoplatin
can bypass these mechanisms and exert a more effective killing of tumor
cells.

Delivery of cisplatin “payload” directly to tumor cells facilitated by DPPG fusion circumventing the
need for Ctr1-receptor mediated transportation required by naked cisplatin. After concentrating in tumors and metastases DPPG promotes the fusion of
Lipoplatin with the cell membrane. Once they reach the tumor target Lipoplatin
nanoparticles have the advantage, unique to Regulon's
technology, to fuse with the cell membrane of the tumor cell and empty their
toxic payload inside the cytoplasm. Liposomes developed by others (e..g. Doxil
of SPI-77 of Alza/J&J) are unable to do the
fusion process; thus the toxic drug is emptied outside the tumor cell and is
less effective.

Import of platins into the cytosol of target cells is highly
regulated to reach energy- and signal (transporter)-dependent biochemical
equilibrium. Shifting this equilibrium in favour of
higher intracellular platinum concentration by means of a suitable drug
delivery system should therefore result in increased apoptosis of target cancer
cells.

Of the various
systems that may achieve acceptable drug delivery in various tissues, liposomal
nanoparticles are gaining ground in targeted cancer therapy, as they help
reducing toxicity of contained therapeutics. The prototype medicinal product
candidate emanating from these efforts is Lipoplatin. Clinical trial results
suggest that PEG coating confers Lipoplatin particles an average circulation
half-life of 40 hours in the plasma of human patients, as opposed to a mere 6
hours for cisplatin. Moreover, once infused into the
patient, Lipoplatin preferentially targets its cytotoxic activity to the
primary and secondary lesions, while leaving normal tissue essentially
unaffected. Extravasation of Lipoplatin is on average 40 times (2 to 200 times
depending on tumor type and vascularisation) more
pronounced in tumors than it is in normal tissues. Moreover, the fusogenic lipid DPPG commands a 5-fold higher fusion of the
Lipoplatin particles to the leaky membrane of the neo-vascularising
tumor cells, as compared to normal cells.

5.4.4. Synergy with radiation for tumor cell killing.Lipoplatin is the only nanoparticle drug
available that contains a heavy metal inside a liposome. Platinum can uptake
high energy from external sources such as laser or gamma rays that can burst
the nanoparticle to release the toxic drug or to heat up the surrounding
cytoplasm. These exciting properties are under current investigation to explore
the full potential of this exciting nanoparticle.

The antiangiogenesis property of Lipoplatin has been suggested
from the encapsulation of the beta-galactosidase gene
into a liposome of the same composition as the Lipoplatin liposome; after
systemic delivery to SCID mice with human tumors (Figure 5) the foreign “blue”
gene stained preferentially the vasculature that the tumors under the skin of
the animals developed to supply the tumor with nutrients. This shows that Regulon's liposomes can target preferentially the vascular
endothelial cells; in case of Lipoplatin, targeting of these cells with toxic cisplatin instead of the “blue” gene would cause their
destruction. Thus, Lipoplatin limits tumor vascularization by attacking their
endothelial cells in addition to the known property of cisplatin
to attack the epithelial cell of the tumor.

The antimetastasis potential of Lipoplatin was shown recently
at the “Reference Oncology Center, Italian National Cancer Institute” in Aviano (http://www.ncbi.nlm.nih.gov/pubmed/24029417).
Lipoplatin inhibited both migration and invasion of
cervical cancer cells supporting its antimetastasis
potential. This is a very important feature of Lipoplatin because migration and
invasion are essential steps used by cancers to mediate their metastases.

5.4.6. Lipoplatin monotherapy (D1,2 200 mg/m2 every 14 days)

A monotherapy
study has shown significant response rate of Lipoplatin in NSCLC mostly applied
as second- and third-line. A partial response of 38% with 43% SD as second-line
chemotherapy is considered significant, rarely seen with other drugs.

A total of 21 patients (2 patients 1st-line, 10 as 2nd-line
and 9 as 3rd-line)

All 21 patients were
evaluable for toxicity. Grade 1 myelotoxicity was
observed in two (9.52%) patients

Grade 1
nausea and vomiting in 4 (19.05%) patients.

Grade 1 fatigue and
peripheral neuropathy in 3 (14.29%) patients.

No alopecia was noted.

During the time of the
drug infusion, temporary myalgia was observed in 5 patients, but it lasted for
only 5-10 min.

Cisplatinmonotherapy induces to patients much higher
toxicities (Grade 3, 4) compared to 0% Grade 3, 4 after Lipoplatin treatment.
In addition, cisplatin displays a much lower efficacy
than Lipoplatin in monotherapy studies (11.1% for cisplatinvs 38.1% for
Lipoplatin). . Cisplatin is considered to be the best
drug or the “Queen of Chemotherapy” among approximately 1,000 oncology products
approved by FDA and EMA. Cisplatin has also a very
broad spectrum against the vast majority of human cancers of epithelial origin
(About 80-90% of cancers are epithelial malignancies). The fact that Lipoplatin
displays such a big difference in efficacy compared to cisplatin
advocates for the value of this drug in cancer management.

Combination of D1,2 Lipoplatin with low-dose
radiation therapy (1 Gy on Day 2) enhances 14 times the radiosensitizing potential of Lipoplatin as shown in
preclinical studies in Canada. Indeed, Lipoplatin was shown to have the best radiosensitizing potential among all platinum compounds. In
these studies, Lipoplatin™ and other platinum
drugs were tested on the F98 glioma cells for their
ability to improve the cell uptake and increase the synergic effect when
combined with ionizing radiation. The cytotoxicity and synergic effect of
platinum compounds were assessed by colony formation assay, while the cellular uptake was measured by Inductively Coupled Plasma Mass
Spectrometer (ICP-MS). After 4 h exposure with platinum compounds, cells
were irradiated (1.5 to 6.6 Gy) with a 60Co source. Lipoplatin compared to cisplatinimproved the cell uptake by 3-fold because of its liposomal nature, and
its radiosensitizing potential was enhanced by
14-fold (Charest et al, 2010).

Regulon proposes a
combination of Lipoplatin monotherapy with low dose
radiation (1-2 Gy). This low dose of radiation should
not cause any side effects to the patients such as burns and tissue damage.
However it will enhance the anticancer effect of Lipoplatin 14 times. Thus, the
already suggested 38:11=3.4-fold higher efficacy of Lipoplatin monotherapy over cisplatinmonotherapy is expected to become 3.4x14 or about 50 fold
higher. This will represent a tremendous medical breakthrough and the success
of such a protocol can be assessed in all human cancers starting with ns-NSCLC,
breast, prostate and stomach cancers. Because it is
not the proper radiation therapy, this low dose can be applied to not only
locally advanced but metastatic cancers.

The total radiation dose in
our proposed protocol is 6-15 Gy for 6 cycles of 14
days plus 9 months maintenance therapy (once every month) for the first year.
Maintenance therapy for the 2nd and 3rd years can be
every 2 and 3 months (6 Gy for the 2nd
year; 4 Gy for 3rd year). Thus, the
patient can be treated with no side effects and an efficacy better than any
other treatment. This could become a universal protocol for all cancers.

6. PHARMACEUTICAL PARTICULARS

6.1. List of lipid excipients

Soy phosphatidyl choline (SPC-3)

Cholesterol

mPEG-DSPE

DPPG

6.2. Incompatibilities

There is a
total loss of cisplatin in 30 minutes at room
temperature when mixed with metoclopramide and sodium metabisulphite
in concentrations equivalent to those that would be found on mixing with a
commercial formulation of metoclopramide. Cisplatin
and sodium bisulphite have been known to react
chemically. Such antioxidants might inactivate cisplatin
before administration if they are present in intravenous fluids.

The same
precaution should be taken for Lipoplatin.

6.3. Shelf life

Prior to first
use: 24 months

In use: 3
months

After
dilution: 7 days under refrigeration

6.4. Special precautions for storage

Lipoplatin is
stored at 2 to 8° C. The vial should never be frozen. Repetitive
freezing and thawing increases the size of the nanoparticles causing formation
of aggregates. Lipoplatin can be shipped at room temperature; there are no
changes in the particle size and other characteristics by storage at room
temperature for up to 20 days. It should be stored at 2 to 8° C upon
arrival to the hospital pharmacy.

6.5. Nature and contents of container

150 mg/50 ml

The 50 ml
clear glass vials of 3 mg/ml (concentration refers to cisplatin)
are closed with rubber closures, and packed as single vials in a small Styrofoam
box.

Packaging:

Lipoplatin
vials will be packaged in cardboard boxes and transported in room temperature.
Upon arrival it should be stored 0°to 4°C.

6.6 Special precautions for disposal and other handling

Refer to local
cytotoxic handling guidelines.

Preparation (Guidelines):

1. Chemotherapeutic agents should be prepared for administration only by
professionals who have been trained in the safe use of the preparation.

2. Operations
such as reconstitution, dilution and transfer to syringes should be carried out
only in the designated area.

3. The
personnel carrying out these procedures should be adequately protected with
clothing, gloves and eye shield.

4. Pregnant
personnel are advised not to handle chemotherapeutic agents

Contamination:

(a) In the
event of contact with the skin or eyes, the affected area should be washed with
copious amounts of water or normal saline. A bland cream may be used to treat
the transient stinging of skin. Medical advice should be sought if the eyes are
affected.

(b) In the
event of spillage, operators should put on gloves and mop up the spilled
material with a sponge kept in the area for that purpose. Rinse the area twice
with water. Put all solutions and sponges into a plastic bag and seal it.

Disposal:

Syringes, container,
absorbent materials, solution and any other contaminated material should be
placed in a thick plastic bag or other impervious container and incinerated.

Administrative Data

7. MARKETING AUTHORISATION HOLDER

Not available
currently. Regulon, Inc. (USA) is the owner of the patent and clinical results.

Represented in
EU by:

Regulon AE

Offices: 7 Afxentiou, 17455 Alimos, Greece

Laboratories R&D: Apollonos
1, 19400 Koropi, Greece

Tel:
+30-210-9858-454

Fax:
+30-210-9858-453

info@regulon.com

www.regulon.com

Licensing and
distribution rights have been assigned to pharmaceutical partners for specific
countries

8. MARKETING AUTHORISATION NUMBER(S)

Will become
available after filing with EMA, London, UK according to the centralized
procedure for the 28 EU countries. Simultaneous or subsequent applications are
expected for US FDA, Taiwanese FDA, Brazilian FDA, Chinese SFDA, Canadian FDA, Australian FDA. Lipoplatin is under registration in Turkey
(2016), Russian Federation (2016), Iran (2016), Libya (2016), while EMA
registration is expected in 2017.

9. DATE OF FIRST AUTHORISATION/RENEWAL OF THE
AUTHORISATION

A request for
Conditional Marketing authorization to the CHMP at EMA was first filed in 2012
for NSCLC (Nanoplatin) and pancreatic cancer
(Lipoplatin). Both filings have been accepted and Rapporteur and co-Rapporteur
have been assigned for the filings by EMA. Meetings between the regulatory team
of Regulon and EMA as well as the Rapporteur took place. Additional data are to
be submitted in 2016 for the final marketing authorisation.